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Methylation of glycosylated sphingolipid modulates membrane lipid topography and pathogenicity
of Cryptococcus neoformans
Arpita Singh1*, Haitao Wang2, Liana C. Silva3, Chongzheng Na2, Manuel Prieto4, Anthony H. Futerman5,
Chiara Luberto1 & Maurizio Del Poeta1,6,7,8
1Biochemistry & Molecular Biology, Medical University of South Carolina, Charleston, South Carolina,
USA; 2Department of Civil Engineering & Geological Sciences, University of Notre Dame, Notre Dame,
Indiana, USA; 3iMed. UL, Faculdade de Farmácia, and 4Centro de Química-Física Molecular, IST,
Lisbon, Portugal; 5Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel;
6Microbiology & Immunology, 7Craniofacial Biology, 8Division of Infectious Diseases, Medical
University of South Carolina, Charleston, South Carolina, USA.
*Present address :- Laboratory of Clinical Infectious Diseases, Molecular Microbiology Section,
National Institute of Allergy and Infectious Diseases, Bethesda, Maryland.
Corresponding Author:
Maurizio Del Poeta, M.D.
Department of Biochemistry and Molecular Biology
Medical University of South Carolina,
173 Ashley Avenue, BSB 512A,
Charleston, South Carolina, 29425
Tel: 843-792-8381; Fax: 843-792-8565; Email: [email protected]
Supplementary materials and methods
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Radiolabelled isotopes. [3H]- palmitic acid and [3H]-myo-inositol are from American Radiolabelled
Chemicals Inc. [32P]- CTP is from Perkin Elmer.
Reconstitution of SMT1 gene. A 3269 bp long fragment containing a 1699 Kb putative SMT1 open
reading frame (ORF), a 1294 bp fragment corresponding to the upstream 5’ untranslated (5’UTR) region
and a 276 bp fragment corresponding to the downstream 3’ untranslated (3’UTR) region was amplified by
PCR using genomic H99 DNA as a template. The 3269 fragment was then cloned in pSC-A-amp/kan
(Stratagene) and sequenced. This fragment was then digested with XbaI, gel purified, eluted and
subcloned in XbaI-digested and dephosphorylated pSK-HYG vector containing the hygromycin B gene
(HYGB conferring resistance to hygromycin B) cassette. HYGB gene is under control of C. neoformans
actin promoter. Hygromycin was purchased from Calbiochem (#400051).
In-vivo [3H]-palmitic acid and [3H]-myo-inositol labeling studies. 15 ml culture, each of WT, ∆smt1 in
YNB supplemented with 25 mM HEPES and 2% glucose, pH 7.2 were grown for 20 hrs in a shaker-
incubator at 37○C in presence of 5% CO2. Approximately 2 hours prior to cell collection, 5x107 cells/ml
were collected and incubated with 2 µCi /ml of [3H]-palmitic acid for 2 hours at the same conditions in a
shaker incubator. For [3H]-myo-inositol labeling, 4 µCi/5x107 cells were used. The cells were then
pelleted, washed in PBS and the lipids were extracted by Mandala and Bligh and Dyer. A quarter of the
sample was aliquoted before drying for the determination of inorganic phosphate. The dried lipid were
dissolved in Chloroform:Methanol (2:1 by volume) before spotting on a TLC and analyzed by radio
scanning. The complex sphingolipid viz IPC, MIPC and M(IP)2C were quantified by scraping the
radiolabelled spots from the TLC. Five µg of soy GlcCer (Avanti Polar Lipids Inc) was loaded as a
control.
In vitro growth studies. A 15 ml starter culture in YPD of WT, Δsmt1 and Δgcs1 from freshly streaked
YPD plates was grown for 24 hrs at 30○C. Cells were pelleted and washed in sterile PBS and counted in a
haemocytometer and resuspended in DMEM supplemented with 50 mM Hepes in pH 7.2 and pH 4.0 to
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have a density of 2.75x105 cells/ml. 200 µl from this suspension were inoculated into a 24 well plate to
have a final cell density of 5.5x104cells/ well and incubated on a shaker at 37○C in presence of 5% CO2 .
Aliquots were taken at the designated time points and serial dilutions were plated on YPD plates for
assessment of colony forming units (CFU).
Tissue burden culture. For tissue burden, organs were homogenized in 10 ml PBS using Stomacher 80
(Lab System Fisher Scientific, Pittsburgh, Pennsylvania, USA) for 2 min at high speed. Serial dilutions
were then plated on YPD plates and incubated for 72 hrs at 30○C. CFU/organ was counted and recorded.
Spot-dilution Assay. Cells from a log phase culture were harvested, washed and resuspended in sterile
PBS at a density of 5x108 cells/ml. 5 µl this cell suspension was spotted in serial dilutions on YPD-agar
plates containing either 0.05% SDS, or 0.025% Triton X-100 or 0.025% Triton X-114 and incubated at
30○C/37○C for 48 hrs or 72 hours in presence of 0.04%/5% CO2. Cell growth was examined and
photographed.
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Supplementary Figure Legends
Figure S1. Illustration of the deletion and the reconstitution scheme of the SMT1 gene (A) The
scheme for the deletion of the SMT1 gene with the deletion cassette p∆smt1. The cassette has the NAT1
gene ORF under C. neoformans actin promoter flanked upstream by 5’UTR and downstream by
3’UTR.The genomic DNA of the homologous recombinants was digested with PstI. Southern Blot
analysis of the transformants with a fragment of 5’UTR, SMT1 ORF and NAT1 as probe showed that the
transformant # 1.2 was formed by a double cross over event with insertion of the NAT1 fragment. It was
designated as C. neoformans ∆smt1. (B) Reconstitution scheme of the SMT1 gene showing the plasmid
cassette with the SMT1 ORF flanked upstream by 5’UTR and downstream by 3’UTR and the HYGB as a
selection marker upstream of 5’UTR. A single cross-over event at the 5’ end resulted in the re-
introduction of the SMT1 gene by the insertion of the plasmid loop and a second copy of the SMT1 gene.
Figure S2. Quantification of the radiolabelled image. The 14C –labelled spot from the in vitro enzyme
assay of the sphingolipid C9 methyltransferase in C. neoformans was scraped off from lane 2 and
quantitated by liquid scintillation counter. Corresponding unlabelled spots from all the other lanes in the
TLC plate were also scraped and quantified. A spot from a blank lane was scraped for individual
background correction. The radio signal was normalized to the corresponding Pi value.
Figure S3. Quantification of complex sphingolipids by in vivo labeling studies with [3H]-Palmitic
acid. WT and ∆smt1 were grown for 20 hours in YNB pH 7.2, 37○C and 5% CO2. Then, 2 µCi of [3H]-
Palmitic acid was added to 5x107 cells for 2 hours prior to cell collection. Cells were harvested by
pelleting and washing in sterile PBS. Lipids were extracted and loaded on a TLC. The radioactive spot
were scraped off, counted in a scintillation counter and the values were normalized with inorganic
phosphate. ∆smt1 shows similar level of complex sphingolipid comprising of IPC, MIPC and M(IP)2C as
compared to the WT. Similar results were obtained using [3H]-myoinositol (data not shown).
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Figure S4. In vitro growth of WT, Δsmt1, Δsmt1+SMT1, Δgcs1, and Δgcs1+GCS1 at pH 7.2 and pH
4.0, 37○C, 5% CO2. Cells from starter culture in YPD were washed and inoculated in DMEM media
supplemented with 50 mM Hepes (either pH 7.2 or pH 4.0) at a concentration of 2.75x105 cells/ml. 200 µl
from this suspension were inoculated in a 24 well plate to have a final cell density of 5.5x104cells/ well
and incubated in a shaker at 37○C, 5% CO2. At different time points, cells in different dilutions were
plated and Colony Forming Units (CFU)/ml were counted after 48-72 h. The ∆smt1 shows a growth
arrest after 36-48 hours at pH 7.2 (A) but not at pH 4.0 (B). (C) A change to acidic medium at pH 4.0
restores growth of both Δsmt1 and Δgcs1 strains.
Figure S5. Tissue burden culture in mice. Brain, spleen, liver, and kidney were collected from CBA/J
mice 90 days post infection with ∆smt1 strain, homogenized in PBS for 2 minutes. Serial dilutions were
plated on YPD plates and incubated for 72 hours at 30○C and CFU/organ was counted.
Figure S6. Histopathology of lung infected with ∆smt1. Lung was excised from CBA/J mice infected
with ∆smt1 cells after 90 days and stained with MOVAT. The picture depicts the ring of foamy
macrophages (white arrows) surrounding necrotic tissue with C. neoformans ∆smt1 cells stained in blue.
Scale white bar: 100 µm.
Figure S7. Histopathology of lung infected with ∆gcs1. A typical granuloma formation in mice lung
infected with ∆gcs1. The nectrotic tissues with several C. neoformans ∆gcs1cells is indicated by the
double arrow. The ring of foamy macrophages is indicated by the white arrow and normal tissue by the
green arrow. Scale white bar: 500 µm.
Figure S8. Spot-dilution assay in presence of SDS. The panels show C. neoformans wild-type (WT)
∆smt1, ∆smt1+SMT1, ∆gcs1, and ∆gcs1+GCS1 spotted in serial dilutions on YPD with 0.05% SDS. The
plates were incubated for 72 hours in presence of 0.04%, 5% CO2, at 30○C.
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Figure S9. A time course of SYTOX GREEN (SG) uptake in untreated cells. C. neoformans WT and
∆smt1 cells grown in YNB, pH 7.2, 37○C and 5% CO2 were pelleted, washed and resuspended in PBS at a
cell density of 108 cell/ml in 24 well plate. 100 µl of PBS + 2 µM SG were added into the wells and
incubated at 37○C in presence of 5% CO2 in dark. Fluorescence was recorded after 20 and 60 minutes of
SG uptake. ∆smt1 cells shows the highest uptake which tends to saturate after 20 minutes. The results are
the mean ± St. Dev. of three separate experiments. Only P<0.05 are shown. ∏P < 0.05, ∆smt1 versus WT.
Figure S10. Illustration of the glucosylceramide pathway in C. neoformans. The de-novo biochemical
pathway of glucosylceramide is shaded. In wild-type cells, only α-OH-Δ4-Δ8, 9methyl-glucosylceramide
is produced. The dotted arrow shows the alternate pathway that Gcs1 takes when the Smt1 or the
sphingolipid desaturase 8 (Sld8) is deleted.
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Table S1 List of restriction enzymes used in this study.
Sl # Primer name
Sequence (5’-3’)
Restriction sites
Purpose
1 Frag 1F CAGGGATCCGAACATATCATCATGGAGCACTTC
BamH1 PCR amplification of 5’-UTR
2 Frag 1R CAGAAGCTTCCACGAAATTGAAATGTTGGAAGT
HindIII PCR amplification of 5’-UTR
3 Frag 3F CGAGATATCGTATGTGCATGTGAATATGATATG
EcoRV PCR amplification of 5’-UTR
4 Frag 3R CAGTCTAGACTAATCTATAAGCAGGCCATCGGT
Xba1 PCR amplification of 5’-UTR
5 R2F CAGTCTAGAGAACATATCATCATGATGGAGCACTTCATC
Xba1 PCR amplification of the C9M gene including the 5’-UTR, ORF and 276 bp of 3’-UTR
6 R2R CAGTCTAGAAGTATCTCCTCCCCGCCAAAGATA
Xba1 PCR amplification of the C9M gene including the 5’-UTR, ORF and 276 bp of 3’-UTR
NA
T15’
UTR
3’U
TR
SM
T15’
UTR
3’U
TR
NA
T15’
UTR
3’U
TR
SM
T15’
UTR
3’U
TRH
YG
NA
T15’
UTR
3’U
TR
NA
T15’
UTR
3’U
TR5’
UTR
SM
T13’
UTR
HY
G
Sal
IS
alI
Sal
I
Sal
I
Sal
IS
alI
Sal
I
Pst
IP
stI
Pst
I
Pst
I
Pst
I
3.26
Kb
5.3
9 Kb
5.0
Kb
3.9
Kb
Pro
be 5
’UTR
Pro
be S
MT1
Pro
be N
AT1
3.0
Kb
5.0
Kb
pSK
-SM
T1-H
YG
pΔsm
t1
WT
Δsm
t1
Δsm
t1
Δsm
t1 +
SM
T1
Sup
plem
enta
ry F
igur
e 1.
Sin
gh e
t al
A B
------
Kb
Sal
I
WT
WT
WT
Δsm
t1Δ
smt1
Δsm
t1
Protein lysate - WT Δsmt1 - WT Δsmt1
Substrate α-OH-Δ4-Δ8-ceramide α-OH-Δ4-Δ8,9methylceramide
cpm
/nm
oles
of P
i
Supplementary Figure 2. Singh et al
Supplementary Figure 3. Singh et al
0
20
40
60
80
100
120cp
m/n
mol
es o
f Pi
IPCMIPC
M(IP)2C
WT ∆smt1
Supplementary Figure 4. Singh et al
0
2
4
6
8
10
0 12 24 36 48 60 720
2
4
6
8
10
0 12 24 36 48 60 72
0
2
4
6
8
10
0 12 24 36 48 60 72 84 96 108 120 132 144
A B
C
Log 1
0 CFU
/ml
Log 1
0 CFU
/ml
Log 1
0 CFU
/ml
Time (h)
pH 7.2 pH 4.0
pH 7.2
Time (h) Time (h)
pH 4.0
WTΔsmt1Δsmt1+SMT1Δgcs1Δgcs1+GCS1
WTΔsmt1Δsmt1+SMT1Δgcs1Δgcs1+GCS1
WTΔsmt1Δsmt1+SMT1Δgcs1Δgcs1+GCS1
Supplementary Figure 5. Singh et al
Organs
Log 1
0 CFU
/org
an
Supplementary Figure 6. Singh et al
Supplementary Figure 7. Singh et al
Supplementary Figure 8. Singh et al
WT∆smt1
∆gcs1
WT
∆smt1+SMT1
∆gcs1+GCS1
8
WT ∆gcs1 ∆smt1
6
4
2
0
20 min
60 min
¥
Supplementary Figure 9. Singh et al
Fluo
resc
ence
uni
t x10
5 /10
8 cel
ls/m
l/sec
∏
Untreated
α-OH-∆4-∆8-Glucosylceramide
α-OH-∆4-∆8, 9Me-Glucosylceramideα-OH-∆4-∆8, 9Me-Ceramide
Gcs1
α-OH-∆4-Δ8-Ceramide
Smt1
α-OH-∆4-Ceramide
Sld8
Gcs1
α-OH-∆4-Glucosylceramide
NH
O
HO
OH
OH
NH
O
HO
OH
OH
NH
HO
OH
OH
O
OO
OH
OH
HOHO
NH
OH
OH
O
OO
OH
OH
HOHO
NH
OH
OH
O
OO
OH
OH
HOHO
NH
OH
OH
O
Gcs1
Supplementary Figure 10 Singh et al
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